aCGH in PGD/PGS

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Comparative genomic hybridization in PGD

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aCGH in PGD/PGS

  1. 1. Comparative genomic hybridization in PGD Dmytro Mykytenko, M.D.,Ph.D. 2012
  2. 2. Embryo aneuploidy• Platteau P., Staessen C.et all, 2004 – 40,5-60% (depends on pathology)• NADIYA (total index) – aCGH – 49,7% – FISH – 59% 2
  3. 3. The main causes of chromosomal anomalies• The inheritance of the parental pathology - true inheritance (e.g.: parental translocation) - Chromosomal nondisjunction during gametogenesis (80-85% of causes relate to oocytes 10-15% - relate to spermatozoa)• Mitotic errors in the zygote 3
  4. 4. Abnormal oocytes Gianaroli, L, Magli, C, et al, Glob. libr. womens med., 2008 4
  5. 5. Preimplantation genetic testing• PDG – is the only one method of detection of embryos without chromosomal / genetic pathology.• The transfer of the embryos without chromosomal imbalances allows us to increase the performance of ART cycles and to prevent the chromosomal pathology of the embryo. Indications to PGS / PGD •Matermal age > 38-42 y.o. •Multiple IVF failure •RPL •High level of sperm aneuploidy •The carriage of the gene defects / chromosomal rearrangements 5
  6. 6. Methods of PGD/PGS• PCR-based – PCR, real-time-PCR – QF-PCR• Sequencing, mini-Sequencing, next-gen sequencing• F.I.S.H.• array-CGH• SNP-array 6
  7. 7. PGD TimeLine PCR -PGD for Fresh ET FISH – sex selection Implementation of aCGH- PGD the FISH into the CGH- PGD aCGH cytogenetics First aCGH-delivery1970 1980 1990 2000 2010 SNP- array PGD First PCR - PGD Implementation of the CGH into the cytogenetics First delivery after aCGH-PGS arrayCGH reported for clinical genetics
  8. 8. What is aCGH?Array comparative genomic hybridization (aCGH) is a technique enabling high-resolution, genomewide screening of segmental genomic copy number variations (M Shinawi, S. W. Cheung. 2008 ) 8 M Shinawi, S. W. Cheung. Drug Discovery Today. 13 (17/18). 2008
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  10. 10. Advantages of the array-CGH• Microscopic chromosomal rearrangements – Aneuploidy (with limited mosaicism detection) – imbalanced rearrangements – Marker chromosomes• Submisroscopic alterations – Subtelomeric imbalanced rearrangements – Micro-deletion/duplication syndromes 10
  11. 11. Disadvantages and limitations• Balanced rearrangements – Reciprocal translocation – Inversions – Robertson translocations – Reciprocal insertions• Imbalanced rearrangements below the diagnostic resolution – Point mutations – Three nucleotide expansions – Deletions / Duplications in not covered regions•Limited ability to detect polyploidy•Limited ability to detect mosaicism•The method needs a great quantity of DNA 11
  12. 12. Whole genome amplification (WGA)• Methods – Primer extension preamplification (PEP) – Degenerative oligonucleotide primed-PCR (DOP-PCR) – Ligation type PCR – Tagget PCR (T-PCR) – Multiple displasement amplification (MDA) – GenpmePlex• Disadvantages – Sensitivity to quality and purity of the input material – Formation of the 100-1000 bp fragments (mean – 400 bp). – Amplification of the 60-80% of the genome only – Effect of the preferential amplification – Secondary DNA structures cause nonspecific amplification – Presence of the active polymerase after the end of the reraction causes subsequent degeneration of the products – Allele drop-out phenomenon not excluded if works with single cell 12
  13. 13. aCGH platforms Technologies: •BAC (1-2 Mbp) •Oligo (10-140kpb) 13
  14. 14. What? Where? and When?Исследуемый материал:• PB 1 & 2• Blastomere (cleavage stage -day 3)• Trophectoderm (blastocyst – day 5) Features PB biopsy Blastomere biopsy TE biopsy •Indirect data about the •Mosaicism is not excluded •More cells = more DNA = more oocyte genotype •Decreasing the embryo accurate diagnostics •Male factor is not taken viability •Less mosaicism into account •Subsequent self-correction of •Reduced impact of embryo biopsy trisomic embryos is not •Economic factor: less embryos to excluded be analized •Facilitates the selective embryo transfer •Allows to modify endometrium if needed •Ability to blastocyst cultivation and vitrification are needed 14
  15. 15. 12h BlueGnome 15
  16. 16. PGD-aCGH (own results) Euploid embryo 46, XY Aneuploid embryo 47, XY, +7 Aneuploid embryoEuploid embryo 45, XY, -1646, XX arr 20q13.32-q.ter x 1 arr CGH 22q11.1-q.ter x 1 16
  17. 17. The comparative analysis of aCGH and FISH PGS (own data) for patients with multiple IVF failure CGH-PGS * FISH-PGS No PGS Cycles total 21 41 134 Main Age 33,9±5,4 33,45±5,1 34,3±4,3 Oocytes retrieved 16,7±8,2 13,6±5,1 12,8±4,2 Previously failed cycles 3,9 ±0,7 3,8±0,6 3,2±0,4 Embryos per ET 1,7±0,5 2,1±1,1 2,6±1,3 ET total 12 (+6 expected) 32 132 Completed cycles 84.4% 78,0% 98,5% Pregnancies total 6 9 18 Pregnancies / OR 40% 22,0% 13,4% Pregnancies / ET 50% 28,1% 13,6% * - frequency of aCGH-PGS cancelation – 34,4% Mykytenko DO, Zukin VD. 1st BRM meeting, 2012 17
  18. 18. Low Y signal 18
  19. 19. Low Y signal 19
  20. 20. aCGH-PGS for different groups of patientsGood Egg donors Young women with good ovarian response Prognosis for ET and pregnancy after aCGH-PGS Couples with male factor Women with age factor Poor ovarian response Carriers of the chromosomal rearrangementsPoor 20
  21. 21. PGD/PGS: aCGH vs other technologiesCriterion aCGH SNP array Next-Gen QF-PCR/ FISH Sequencing PCR PGSComprehensive + + + - -chrom. screeningBalanced - - ? - LimitedaberrationsNonbalanced + + + + +abberationsMicrodeletion Limited + + + +syndromesUPD - + + - -Single Gene - +/- + + -disorders 21
  22. 22. Future directions• arrayCGH will compete with SNP-arrays and next- gen. sequencing-based methods in the range of the same indications to testing• FISH-method will not die out (at least in close and middle future) due to different indication to testing• Further investigations will be directed to: •comparison of the arrayCGH results with embryo morphokinetics and ‘–omics’ characteristics •development of the accurate criterions for the selective ET 22
  23. 23. Conclusions• PGS/PGD testing allows us to increase the performance of ART technologies and to reduce the amount of unsuccessful ET.• Among all similar PGS-methods, array-Comparative Gnomic Hybridization seems to be the most suitable approach to detect the embryo aneuploidy.• Performing of aCGH-PGS with the number of embryos less than 3 is inappropriate.• aCGH should not be the cause of ungrounded rejection of the normal embryo! 23
  24. 24. It’s hard to being a good embryo But even more difficult to detect it… d.mykytenko@genetics.kiev.ua 24

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